Project Summary: Modifications of N-glycans (oligosaccharides N-linked to an extracellular Asn residue in a membrane protein) are associated with neurological difficulties as emphasized by congenital disorders of glycosylation. There are three general types of N-glycans: oligomannose, hybrid and complex. Further the hybrid and complex types can have bisecting type N-glycans which are quite common in brain. These various N-glycans differ by the number of branches attached to their conserved pentasaccharide. To date, studies have shown the importance of occupancy of N-glycosylation sites for the function of neuronal voltage gated K+ channel (Kv) channels. However, the relevance of the structure of N-glycans attached to Kv channels is unknown. Our lab has previously shown that N-glycan site occupancy regulates the Kv3.1b channel, a critical component of fast-spiking neurons, in neuroblastoma cells and adult primary neurons, and more recently, we have expanded our studies to reveal that substitution of complex type N-glycans with hybrid type reduces the level of Kv3.1b protein to the neurites, and slows the opening and closing rates of the Kv3.1b channel. Our long-term goal is to investigate how modifications in N-glycosylation processing can modulate the structure and function of neurons since these discoveries will enhance the design of therapeutics for human neurodegenerative diseases. Our central hypothesis is that changes in branched N-glycan structures attached to the Kv3.1b voltage gated K+ channel can alter neural excitability and organismal behavior. To test our hypothesis, we will investigate if change in branching of N- glycan structures modifies the excitability of Kv3.1b-expressing neuroblastoma cells (aim 1). Additionally, we will explore if unoccupied N-glycosylation sites of the Kv3.1b channel perturbs excitability and development of fast- spiking neurons and thus locomotion in zebrafish (aim 2). This innovative proposal employs a simplified approach, along with numerous levels of analysis, to explore the roles of various branched N-glycans, and to establish N-glycosylation as mechanism for modulating Kv3.1b function in vivo. In conclusion, our study will provide fundamental knowledge to better understand human neurological diseases, thereby potentially introducing improved treatment options.